In many respects, the zebrafish surpasses the mouse in utility as a model organism for understanding human embryonic development and disease. Zebrafish embryos are optically clear and easily manipulated, permitting sub-cellular biomedical imaging studies in living tissue. Moreover, small molecule compounds can be easily applied to zebrafish larvae, allowing screens for novel therapeutic agents that are capable of suppressing mutant phenotypes. What is lacking, however, are homologous recombination technologies for targeting specific (and clinically relevant) mutations into the zebrafish genome (for use in these studies). The specific aim of this proposal is to develop gene-targeting technology for zebrafish, by adapting an in vivo transgene-based homologous recombination (HR) strategy that has been successfully used in Drosophila. The strategy involves three components: 1) a targeting vector that is integrated into the genome as a transgene, and flanked by recombinase and endonuclease recognition sites;2) an inducible recombinase enzyme that will excise the transgene from the genome as a closed circular extra- chromosomal vector;and 3) an endonuclease activity that will generate DNA double-strand breaks (DSBs) in the targeting vector, rendering it recombinogenic. In Drosophila studies, this nuclear DNA fragment can efficiently recombine with homologous loci. We hypothesize that a similar approach can be used to mutate the zebrafish genome. To this end, we will develop the following technological components and strategies: A) Targeting vector and selection. As a proof of principal, we will first target vasa, a germ-line specific gene. In preliminary studies, we have designed the targeting vector such that recombination into the vasa locus will specifically drive GFP expression in germ cells. This will allow direct and efficient screening for heritable homologous recombination events. B) Liberation of targeting vector: In a first approach, directly based on the Drosophila methodology, we will utilize Cre recombinase to excise targeting vectors from the genome. We will then attempt to streamline the gene targeting strategy by utilizing Tol2 transposase activity to mobilize the vasa-GFP targeting vector. C) Generation of DSBs: In preliminary studies we have identified two mega-endonucleases that are functional in zebrafish embryos. We propose to utilize these enzymes to specifically generate DSBs in extra-chromosomal targeting vectors, in vivo. D) Timing of recombination: We will utilize an oocyte-specific promoter element to express endonuclease and recombinase activities during meiotic prophase 1, when the oocyte is primed for homologous recombination. This will maximize the efficiency of gene targeting. F0 fish that harbor i) targeting vector, ii) recombinase, and iii) endonuclease transgenic elements will be generated. Gene targeting events will initiate in the ovary, and will be detected by screening F1 progeny for germ cell-specific GFP expression. PUBLIC HEALTH RELEVANCE: Homologous recombination technologies, as developed in this proposal, will allow researchers to specifically mutate and manipulate the zebrafish genome. This will allow geneticists to I) target clinically relevant mutations into zebrafish, to mimic human birth defects and disease);and 2) generate "conditional" zebrafish mutants so that gene function can be studied in specific tissues or cell lineages, or at different stages of development. Using these new resources, researchers will be able to exploit the experimental advantages of zebrafish to gain new insights into the molecular and genetic mechanisms that control embryonic development, behavior and disease, and to discover novel therapeutic agents that can suppress mutant phenotypes and treat human disease.